1. Technical Field
The presently disclosed invention embodiments relate to RNA nanoparticles and methods for their preparation and use. In particular, the present embodiments relate to multiple-way junctional fragments of dsDNA virus packaging RNA (pRNA), and their derivatives, for assembling and stabilizing multivalent RNA nanoparticles.
2. Description of the Related Art
In living systems there exists a wide variety of highly-ordered or patterned structures including smart nanomachines, and elegant arrays that are made up of macromolecules to perform diverse biological functions. Over the past 30 years, for instance, there has been a rapid expansion of knowledge in DNA nanotechnology. Both RNA and DNA share some common properties, such as polynucleotide strand complementarities and self-assembly, and can serve as powerful building blocks for bottom-up fabrication of nanostructures and nano-devices.
There has been a heightened interest in RNA therapeutics since the discovery of small interfering RNA (siRNA). RNA interference (RNAi) is a polynucleotide sequence-specific, post-transcriptional gene silencing mechanism effected by double-stranded RNA that results in degradation of a specific messenger RNA (mRNA), thereby reducing the expression of a desired target polypeptide encoded by the mRNA (see, e.g., WO 99/32619; WO 01/75164; U.S. Pat. No. 6,506,559; Fire et al., Nature 391:806-11 (1998); Sharp, Genes Dev. 13:139-41 (1999); Elbashir et al. Nature 411:494-98 (2001); Harborth et al., J. Cell Sci. 114:4557-65 (2001)). RNAi is mediated by double-stranded polynucleotides as also described hereinbelow, for example, double-stranded RNA (dsRNA), having sequences that correspond to exonic sequences encoding portions of the polypeptides for which expression is compromised. RNAi reportedly is not effected by double-stranded RNA polynucleotides that share sequence identity with intronic or promoter sequences (Elbashir et al., 2001). RNAi pathways have been best characterized in Drosophila and Caenorhabditis elegans, but “small interfering RNA” (siRNA) polynucleotides that interfere with expression of specific polypeptides in higher eukaryotes such as mammals (including humans) have also been described (e.g., Tuschl, 2001 Chembiochem. 2:239-245; Sharp, 2001 Genes Dev. 15:485; Bernstein et al., 2001 RNA 7:1509; Zamore, 2002 Science 296:1265; Plasterk, 2002 Science 296:1263; Zamore 2001 Nat. Struct. Biol. 8:746; Matzke et al., 2001 Science 293:1080; Scadden et al., 2001 EMBO Rep. 2:1107) and subsequently elaborated upon.
According to a current non-limiting model, the RNAi pathway is initiated by ATP-dependent, processive cleavage of long dsRNA into double-stranded fragments of about 18-27 (e.g., 19, 20, 21, 22, 23, 24, 25, 26, etc.) nucleotide base pairs in length, called small interfering RNAs (siRNAs) (see review by Hutvagner et al., Curr. Opin. Gen. Dev. 12:225-32 (2002); Elbashir et al., 2001; Nykänen et al., Cell 107:309-21 (2001); Zamore et al., Cell 101:25-33 (2000); Bass, Cell 101:235-38 (2000)). In Drosophila, an enzyme known as “Dicer” cleaves the longer double-stranded RNA into siRNAs; Dicer belongs to the RNase II family of dsRNA-specific endonucleases (WO 01/68836; Bernstein et al., Nature 409:363-66 (2001)). Further according to this non-limiting model, the siRNA duplexes are incorporated into a protein complex, followed by ATP-dependent unwinding of the siRNA, which then generates an active RNA-induced silencing complex (RISC) (WO 01/68836). The complex recognizes and cleaves a target RNA that is complementary to the guide strand of the siRNA, thus interfering with expression of a specific protein (Hutvagner et al., supra).
In C. elegans and Drosophila, RNAi may be mediated by long double-stranded RNA polynucleotides (WO 99/32619; WO 01/75164; Fire et al., 1998; Clemens et al., Proc. Natl. Acad. Sci. USA 97:6499-6503 (2000); Kisielow et al., Biochem. J. 363:1-5 (2002); see also WO 01/92513 (RNAi-mediated silencing in yeast)). In mammalian cells, however, transfection with long dsRNA polynucleotides (i.e., greater than 30 base pairs) leads to activation of a non-specific sequence response that globally blocks the initiation of protein synthesis and causes mRNA degradation (Bass, Nature 411:428-29 (2001)). Transfection of human and other mammalian cells with double-stranded RNAs of about 18-27 nucleotide base pairs in length interferes in a sequence-specific manner with expression of particular polypeptides encoded by messenger RNAs (mRNA) containing corresponding nucleotide sequences (WO 01/75164; Elbashir et al., 2001; Elbashir et al., Genes Dev. 15:188-200 (2001)); Harborth et al., J. Cell Sci. 114:4557-65 (2001); Carthew et al., Curr. Opin. Cell Biol. 13:244-48 (2001); Mailand et al., Nature Cell Biol. Advance Online Publication (Mar. 18, 2002); Mailand et al. 2002 Nature Cell Biol. 4:317).
siRNA polynucleotides may offer certain advantages over other polynucleotides known to the art for use in sequence-specific alteration or modulation of gene expression to yield altered levels of an encoded polypeptide product. These advantages include lower effective siRNA polynucleotide concentrations, enhanced siRNA polynucleotide stability, and shorter siRNA polynucleotide oligonucleotide lengths relative to such other polynucleotides (e.g., antisense, ribozyme or triplex polynucleotides).
By way of a brief background. “antisense” polynucleotides bind in a sequence-specific manner to target nucleic acids, such as mRNA or DNA, to prevent transcription of DNA or translation of the mRNA (see, e.g., U.S. Pat. No. 5,168,053; U.S. Pat. No. 5,190,931; U.S. Pat. No. 5,135,917, U.S. Pat. No. 5,087,617; see also, e.g., Clusel et al., 1993 Nucl. Acids Res. 21:3405-11, describing “dumbbell” antisense oligonucleotides). “Ribozyme” polynucleotides can be targeted to any RNA transcript and are capable of catalytically cleaving such transcripts, thus impairing translation of mRNA (see, e.g., U.S. Pat. No. 5,272,262; U.S. Pat. No. 5,144,019; and U.S. Pat. Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246; U.S. 2002/193579). “Triplex” DNA molecules refers to single DNA strands that bind duplex DNA to form a colinear triplex molecule, thereby preventing transcription (see. e.g., U.S. Pat. No. 5,176,996, describing methods for making synthetic oligonucleotides that bind to target sites on duplex DNA). Such triple-stranded structures are unstable and form only transiently under physiological conditions.
Because single-stranded polynucleotides do not readily diffuse into cells and are therefore susceptible to nuclease digestion, development of single-stranded DNA for antisense or triplex technologies often requires chemically modified nucleotides to improve stability and absorption by cells. siRNAs, by contrast, are readily taken up by intact cells, are effective at interfering with the expression of specific polypeptides at concentrations that are several orders of magnitude lower than those required for either antisense or ribozyme polynucleotides, and do not require the use of chemically modified nucleotides.
RNA-based therapeutic approaches using siRNA, ribozymes and anti-sense RNA have been shown to down-regulate specific gene expression in cancerous or viral-infected cells. RNA has therefore been particularly attractive as a therapeutic platform, since it can be manipulated with simplicity characteristic of DNA, while possessing non-canonical base-pairing, versatile function and catalytic activity similar to that of proteins. Typically, RNA molecules contain a large variety of single-stranded stem-loops for inter- and/or intra-molecular interactions. These loops can serve as mounting dovetails, and thus external linking dowels might not be needed in fabrication and assembly.
Although the concept of RNA nanotechnology has been developed for more than ten years, the popularity of studying RNA nanostructures has emerged recently, as reflected by the observation that 90% of publications on RNA nanostructures were published during or after 2006. For example, the DNA packaging motor of the double-stranded DNA (dsDNA) bacteriophage phi29 is geared by a hexameric packaging RNA (pRNA) ring. Each pRNA contains two functional domains. The central domain of each pRNA subunit contains two interlocking loops, denoted as the right- and left-hand loops that can be reengineered to form dimers, trimers, tetramer, pentamer, hexamers or heptamer via hand-in-hand interactions (FIG. 3). The helical DNA packaging domain is located at the 5′/3′ paired ends. The two domains fold separately, and replacement of the helical domain with a siRNA (or with a ribozyme or an antisense RNA) does not affect pRNA structure, folding or intermolecular interactions. The resultant pRNA/siRNA chimera has been demonstrated to be useful for gene therapy. In addition, the 5′/3′ paired helical region serves as a site for the binding of a DNA packaging enzyme gp16.
RNA is therefore a particularly attractive building block for bottom-up fabrication of nanostructures. In a number of described systems, self-assembled nanoparticles composed of multiple RNA building blocks have been used as vehicles to escort siRNA, ribozymes, antisense RNA or other therapeutics to specific cells.
One of the challenges in this emerging field, however, is the relative instability of RNA nanoparticles that are formed without covalent modifications or chemical cross-linking, resulting in their dissociation at ultra low concentrations in vivo in human and animal circulation systems. This instability has seriously hindered the delivery efficiency and therapeutic applications of RNA nanoparticles. In addition, magnesium is critical for RNA folding and the optimum concentration is in the range of tens of mM; however, under physiological conditions (e.g., in the circulation of humans and other mammals), the magnesium concentration is less than 1 mM. Such low magnesium concentrations could result in RNA misfolding, as well as dissociation of the RNA nanostructures. An obstacle to reliable and convenient systemic delivery of therapeutic RNA nanoparticles is thus their dissociation at low concentrations, along with misfolding or unfolding in the low-magnesium environment after entering the circulatory system in the body.
Clearly there remains a need in the art for improved compositions and methods for preparation and delivery of nanoparticles such as therapeutic RNA-based nanoparticles. The herein described invention embodiments address this need and provide other related advantages.